The overall goal of this project is to unravel the molecular determinants linking sympathetic nerve stimulation (SNS) and regulation of the IKS (KCNE1/KCNQ1) channel. SNS activates beta adrenergic receptors (beta-ARs), increases cAMP, and PKA phosphorylates the IKS (KCNE1/KCNQ1) channel to increase repolarizing current and shorten the cardiac action potential action potential duration (APD) in the face of a concomitant increase in heart rate. PKA regulation of the IKs channel requires assembly of a macromolecular signaling complex that is coordinated by the binding of the targeting protein yotiao to a leucine zipper motif in the KCNQ1 carboxy-terminal (C-T) domain. Dysfunction in this regulatory pathway likely triggers fatal arrhythmias in two variants of the Long QT syndrome (LQTS). The proposed work focuses on novel inter- and intra-molecular interactions within this complex that are required to regulate this important ion channel and on mechanisms by which disruption of this regulation may lead to electrical instability. There are four specific aims: Aim 1 is to test the hypothesis that inter-molecular interactions between the IKs channel beta subunit (KCNE1) carboxy-terminal (C-T) domain and the alpha subunit (KCNQ1) amino terminal (N-T) domain are required for PKA-induced changes in IKs activity; Aim 2 is to test the hypothesis that PKA phosphorylation of the adaptor protein Yotiao is necessary for SNS modulation of IKs channels; Aim 3 is to test the hypothesis that intramolecular interactions between the KCNQ1 N-T and C-T domains are required for modulation of IKS channels by SNS; and Aim 4 is to use in vivo and in silico models to test the hypothesis that disruption of IKs regulation by SNS can underlie unstable cellular electrical activity and that may trigger arrhythmias. The planned experimental approach combines molecular biology, biochemistry, functional measurements using patch clamp electrophysiology, genetically-altered mice, and computational modeling of ion channel activity. The results of these investigations will provide novel insight into and understanding of the molecular interactions that underlie this fundamental and essential cardiac response.